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Abstract

Transforming growth factor β1 (TGFβ1) is a cytokine with multiple functions. TGFβ1 significantly induces migration and invasion of liver cancer cells. However, the molecular mechanisms underlying this effect remain unclear. Epithelial‑to‑mesenchymal transition (EMT) is crucial for the development of invasion and metastasis in human cancers. The aim of the present study was to determine whether TGFβ1‑induced EMT promoted migration and invasion in HepG2 cells. The underlying mechanism and the effect of EMT on HepG2 cells were also investigated. The results demonstrated that TGFβ1 may induce EMT to promote migration and invasion of HepG2 cells, and this effect depends on activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway. JAK/STAT3 signaling is involved in human malignancies, including lung cancer, and is implicated in cell transformation, tumorigenicity, EMT and metastasis. In the present study, TGFβ1 also activated JAK/STAT3 signaling in HepG2 cells and promoted Twist expression, but these events were abolished by treatment with the STAT3 inhibitor AG490. Additionally, Twist siRNA blocked TGFβ1‑induced EMT. Thus, TGFβ1 was shown to induce EMT, thereby promoting the migration and invasion of HepG2 cells via JAK/STAT3/Twist signaling.

Introduction

Liver cancer is one of the most common malignant
tumors worldwide, and its incidence is the second highest in China.
Liver cancer cannot be easily detected at an early stage due to the
lack of distinct symptoms and the scarcity of clinically specific
markers for serodiagnosis. Therefore, the majority of the patients
are diagnosed at advanced or late stages, resulting in distant
metastasis and a low 5-year survival rate (1). Thus, the metastasis and invasion of
liver cancer must be clinically investigated to prevent progression
of this disease and improve its prognosis.

The invasion and metastasis of malignant tumors are
regulated and controlled by various factors and mechanisms.
Epithelial-to-mesenchymal transition (EMT) is a key mechanism
participating in the invasion and metastasis of solid cancers, such
as colon, lung and pancreatic cancer (2–4).
However, the association between EMT and the onset and progression
of liver cancer has not been fully elucidated. In this context, Lee
et al (5) and Giannelli
et al (6) previously
reported that EMT is involved in the invasion and metastasis of
liver cancer cells.

A number of studies reported that transforming
growth factor β1 (TGFβ1) is a cytokine with multiple functions that
promotes EMT (7,8). The activation abnormalities in the
signal transducer and activator of transcription 3 (STAT3)
signaling pathway are associated with tumor onset and progression
(9). The activation of this
pathway is regulated and controlled by the upstream factor Janus
kinase (JAK). The activation of JAK/STAT3 signaling may directly
affects EMT and promotes the invasion and metastasis of tumor cells
in lung cancer and ovarian tumors (10). However, whether the EMT mediated
by the JAK/STAT3 signaling pathway promotes TGFβ1-induced invasion
and metastasis of liver cancer cells has not been clearly
determined.

The present study investigated the human liver
cancer line HepG2, in which invasion and metastasis were induced by
TGFβ1. The role of JAK/STAT3 signaling in mediating the involvement
of EMT in the invasion and metastasis of HepG2 cells induced by
TGFβ1 was also determined. Experiments were performed to confirm
whether Twist is a target of STAT3. Overall, the aim of this study
was to provide new experimental evidence and potential targets for
preventing the invasion and metastasis of liver cancer cells.

RNA was extracted from the tissue samples using
TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA,
USA), according to the manufacturer's instructions. Subsequently,
cDNA was synthesized using a TaqMan Reverse Transcription Reagents
kit (Thermo Fisher Scientific), according to the manufacturer's
protocol. The relative expression levels of mRNA were determined
using a Power SYBR-Green PCR Master Mix kit (Thermo Fisher
Scientific) and normalized to GAPDH. RT-PCR was performed using the
Applied Biosystems 7500 Fast Dx Real-Time PCR instrument (cat no.
4425757; Thermo Fisher Scientific) and the following gene-specific
primers (Sangon Biotech Co., Ltd., Shanghai, China): GAPDH: Sense,
5′-TGCCATCAACGACCCCTTCA-3′ and antisense,
5′-TGACCTTGCCCACAGCCTTG-3′; E-cadherin: Sense,
5′-AGCTATCCTTGCACCTCAGC-3′ and antisense, 5′-CCCAGGAGTTTGAG-3′;
N-cadherin: Sense, 5′-TCCTGCTCACCACCACTACTT-3′ and antisense,
5′-CTGACAATGACCCCACAGC-3′; Smad: Sense, 5′-ATAAGCAACCGCCTGAACAT-3′
and anti-sense, 5′-TTACCTGCCTCCTGAAGACC-3′; Twist: Sense,
5′-GCTGATTGGCACGACCTCT-3′ and antisense,
5′-CACCATCCTCACACCTCTGC-3′; and vimentin: Sense,
5′-CCAAACTTTTCCTCCCTGAACC-3′ and antisense,
5′-GTGATGCTGAGAAGTTTCGTTGA-3′. A control siRNA specific for the red
fluorescent protein, 5′-CCACTACCTGAGCACCCAG-3′, was used as the
negative control (sc-37007; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA). All primers were designed using the National Center
for Biotechnology Information Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR
was performed under the following conditions: Denaturation at 50°C
for 2 min, followed by 38 cycles at 95°C for 15 sec and 60°C for 1
min. Gene expression was normalized to internal controls and fold
changes were calculated using the relative quantification method
(2−ΔΔCq) (11).

Scattering assay

Scattering assay was performed as previously
described (7). HepG2 cells
(3×105/ml) were seeded into each well of a 24-well plate
(cat. no. 662102; Greiner Bio-One GmbH, Frickenhausen, Germany),
and incubated overnight at 37°C in an atmosphere of 5%
CO2. The cells were pretreated with 10 μM TGFβ1
for 48 h at 37°C for 48 h in 95% air and 5% CO2.
Representative images were captured at a magnification of ×20 using
the Eclipse TE2000-U inverted microscope (Nikon Corporation, Tokyo,
Japan).

Invasion and migration assay

The invasion assay was performed using Transwell
24-well plates with 8-μm pore polycarbonate membranes (BD
Biosciences, Franklin Lakes, NJ, USA). Briefly, the upper side of
the membranes was coated with Matrigel (20 μg/well) and the
membranes were then air-dried for 1 h at 37°C. The lower side of
the membranes was coated with 5 μg fibronectin, and the
treated or untreated HepG2 cells (2×105) in 200
μl of DMEM medium with 2.5% FBS were placed in the upper
chamber. The lower chamber was filled with DMEM medium with 10% FBS
as the chemoattractant. The invasion chamber was incubated for 8 h
at 37°C and 5% CO2. The cells on the upper surface of
the membrane were removed by gentle scrubbing with a cotton swab.
The membranes were fixed in a stationary liquid of 95% ethanol and
5% acetic acid for 30 min and stained with crystal violet. The
number of cells on the lower surface of the membrane in 5 random
visual fields (magnification, ×200) was then counted using an
Eclipse TE2000-U inverted microscope. Each assay was performed in
triplicate.

Wound healing assay

The wound healing assay was performed as previously
described: HepG2 cells (3×105/ml) were seeded into a
6-well plate (cat. no. 657160; Greiner Bio-One GmbH) in
serum-containing medium, and incubated at 37°C in an atmosphere of
5% CO2 in order to form a confluent monolayer. The
monolayer was scratched using a sterile plastic pipette tip (cat.
no. CLS4860; Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA), and
washed with PBS to remove cell debris. Subsequently, fresh medium
was added, and 10 μM TGFβ1 or 0.1 ml DMSO was added to each
well. The scratched mono-layer was incubated at 37°C in an
atmosphere of 5% CO2 for 48 h. Wound closure was
measured in 6 random high-power fields at a magnification of ×200,
using Image-Pro®Express software, version 6 (Media
Cybernetics, Inc., Rockville, MD, USA) and an Eclipse TE2000-U
inverted microscope (11).

Statistical analysis

Data were analyzed using SPSS software, version 1.0
(SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software, version
5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Analysis of
variance was conducted followed by the Student's t-test. The data
are presented as mean ± standard deviation. P<0.05 was
considered to indicate statistically significant differences.

Results

TGFβ1 induces migration and invasion of
HepG2 cells

To determine the migration and invasion induced by
TGFβ1, liver cancer HepG2 cells were treated with different
concentrations of TGFβ1 for 48 h, and the migration and invasion of
cancer cells were assessed by wound closure assays and Matrigel
Transwell chamber invasion assays. The effects of TGFβ1 were
observed at concentrations as low as 5 μM. As the TGFβ1
concentration increased, the migration and invasion of HepG2 cells
also increased in a concentration-dependent manner, with the most
prominent effects observed at a concentration of 10 μM
(Fig. 1A and B). These results
indicated that TGFβ1 induced HepG2 cell migration and invasion in a
concentration-dependent manner. Hence, the concentration of 10
μM was selected for all further mechanistic studies.

TGFβ1 induces EMT in HepG2 cells

TGFβ1 is a factor that promotes EMT in cancer cells,
as previously reported (7,8).
EMT is an important mechanism of cancer cell invasion and
metastasis. The downregulation of E-cadherin expression and the
upregulation of vimentin and N-cadherin expression are considered
to be markers of EMT. In the present study, TGFβ1 also induced cell
scattering (Fig. 2A), indicating
that TGFβ1 induces EMT, thereby increasing the migration and
invasion of HepG2 cells. To further investigate whether EMT is
involved in TGFβ1-induced scattering, migration and invasion of
HepG2 cells, the expression of E-cadherin, vimentin and N-cadherin
were first detected by qPCR and western blot analysis. As shown in
Fig. 2B and C, the expression of
vimentin and N-cadherin was upregulated, whereas that of E-cadherin
was downregulated following treatment with 10 μM TGFβ1.
These results demonstrated that TGFβ1-induced EMT promoted the
migration and invasion of HepG2 cells.

Moreover, JAK/STAT3 protein expression was detected
by western blot analysis and it was observed that TGFβ1 stimulated
the expression of p-JAK and p-STAT3. This finding indicates that
TGFβ1-induced EMT may be activated by JAK/STAT3 signaling.

JAK/STAT3 signaling is involved in
TGFβ1-induced EMT to increase migration and invasion of HepG2
cells

STAT3 is the key transcription factor regulating
cell proliferation and survival. STAT3 may be activated by
oncostatin M, interferons, interleukin-6 (IL-6) and epidermal
growth factor (EGF). It was recently reported that TGFβ1 induced
JAK/STAT3 signaling to increase migration and invasion in lung
carcinoma cells. Based on these reports, we hypothesized that
JAK/STAT3 signaling may be involved in TGFβ1-induced EMT to
increase the migration and invasion in HepG2 cells. To confirm this
hypothesis, HepG2 cells were incubated with the STAT3 inhibitor
AG490 prior to treatment with TGFβ1. As shown in Fig. 3A and C, AG490 significantly
suppressed the TGFβ1-induced upregulation of N-cadherin and
vimentin expression and the downregulation of E-cadherin
expression, compared with the TGFβ1 group. Furthermore, AG490
treatment significantly reduced the number of TGFβ1-induced
invasive and migratory cells (Fig. 3E
and F), which was consistent with the results obtained from
metastasized cell-wound closure (Fig.
3D).

Twist is involved in TGFβ1-induced EMT
depending on STAT3

Smad2 and Twist are important factors regulating EMT
through TGFβ1. Thus, we hypothesized that TGFβ1 induced EMT by
upregulating Twist expression via JAK/STAT3 signaling. To confirm
this hypothesis, the expression of pSmad2 and Twist in HepG2 cells
treated with TGFβ1 was first detected. The results revealed that
TGFβ1 induced the protein expression of pSmad2 and Twist (Fig. 4A–D). By contrast, AG490 treatment
reversed the TGFβ1-induced protein expression of pSmad2 and Twist
(Fig. 4E–G). Along these lines,
TGFβ1 induced the protein expression of pSmad2 and Twist in
accordance with the activated JAK/STAT3 signaling.

To further determine whether Twist participated in
TGFβ1-induced EMT, HepG2 cells were transfected with siRNA of
Twist. As shown in Fig. 5A–C,
Twist knockdown significantly suppressed the TGFβ1-induced
expression of N-cadherin and vimentin, but reversed the
TGFβ1-inhibited expression of E-cadherin. The TGFβ1-induced
migration and invasion in HepG2 cells were also reversed through
Twist knockdown. Overall, these data strongly suggest that Twist
participates in TGFβ1-induced EMT to increase the migration and
invasion of HepG2 cells via JAK/STAT3 signaling.

Discussion

Invasion and metastasis are the leading causes of
death from liver cancer (12).
The onset and progression of liver cancer are regulated and
controlled by various factors, among which EMT is the key mechanism
promoting invasion and metastasis (13). A number of studies demonstrated
that molecular markers are altered during EMT in cancer cells; for
example, E-cadherin (an epithelial marker) and ZO-1 (a closely
connected protein) are downregulated, whereas the levels of
molecular markers derived from interstitial cells, including
vimentin and N-cadherin, are upregulated. Hence, adhesions among
tumor cells are reduced, thereby increasing the invasion and
metastasis of cancer cells (14–16). The occurrence of EMT is affected
by various factors. TGFβ1 is a key factor that induces and
participates in the entire process of EMT (17–19). In the present study, TGFβ1 was
found to upregulate vimentin and downregulate E-cadherin
expression. Moreover, TGFβ1 induced scattering, invasion and
metastasis of HepG2 cells. These results demonstrated that
TGFβ1-induced EMT, thereby promoting the invasion and metastasis of
HepG2 cells.

STAT3 is a signal transduction and transcription
activator. Abnormal regulation of the STAT3 signaling pathway is
associated with tumor occurrence and development (20). Following activation by cytokines
or growth factors, the activated JAK may collect STAT3 monomers to
produce homologous or heterogonous dimers; subsequently, nuclei and
specific DNA sequences regulate the transcription of target genes
(21). The abnormal expression
and activation of STAT3 in various tumor tissues and cell lines
(including liver cancer cells) are associated with the invasion and
metastasis of tumor cells (21).
EMT is the first focus in studies investigating the invasion and
metastasis of tumor cells. The activation of the STAT3 signaling
pathway is associated with EMT, invasion and metastasis of tumors.
Colomiere et al (22)
demonstrated that the JAK̸STAT3 pathway is aberrantly activated in
ovarian cancer tissues. Furthermore, EMT in ovarian cancer cells
may be induced by EGF or IL-6 (23,24). These results indicated that the
action of EGF or IL-6 relies on the activation of JAK̸STAT3
signaling; EMT induced by EGF or IL-6 may be significantly
inhibited by treatment with the JAK̸STAT3 pathway inhibitor AG490,
and the invasion and metastasis of ovarian cancer cells may be
reduced. Xiong et al (25)
also reported that AG490 significantly suppressed STAT3 activation;
consequently, AG490 treatment upregulated E-cadherin expression but
reduced the invasion of tumor cells in colorectal cancer. In the
present study, the JAK/STAT3 pathway was activated when the
TGFβ1-induced EMT promoted the migration and invasion of HepG2
cells, whereas AG490 reversed these effects. Overall, the results
demonstrated that TGFβ1-induced EMT was inhibited, thereby
confirming the involvement of the JAK/STAT3 pathway in EMT
induction.

Lee et al (7) reported that JAK̸STAT3 pathway
activation promotes the expression of Twist and, thus, reduces the
EMT of breast cancer cells. Cheng et al (17) also reported that the activated
STAT3 may directly bind to the STAT3 binding site of the Twist
promoter in breast cancer cells. These studies support that STAT3
activation may regulate the expression of Twist, a key
transcription factor regulating EMT. In the present study, Twist
was downstream from the JAK/STAT3 pathway in HepG2 cells; thus,
AG490 treatment was applied for 2 h prior to incubation with TGFβ1.
Our results demonstrated that the expression of Twist was inhibited
by AG490. Overall, TGFβ1 induced the expression of Twist in
accordance with the JAK/STAT3 pathway activation. Furthermore, we
observed that Twist participated in the TGFβ1-induced EMT that
promoted invasion and metastasis in HepG2 cells. This finding is
consistent with the report of Liu et al (26). Thus, these results indicated that
TGFβ1 upregulated the expression of Twist via the JAK/STAT3
pathway, thereby promoting the invasion and metastasis of HepG2
cells.

The findings of the present study verified the
biological functions of TGFβ1 in liver cancer HepG2 cells and
provided evidence that the TGFβ1-induced EMT promoted the invasion
and metastasis of HepG2 cells in vitro. It was further
demonstrated that these actions may be mediated via the
JAK/STAT3/Twist signaling pathway. In conclusion, TGFβ1 appears to
be involved in the progression of liver cancer and represents a
potential molecular target for the treatment of this disease.

Acknowledgments

This study was supported by the Natural Science
Foundation of China (grant no. 81600342), the Medical Foundation of
Hui Zhou (grant no. 2015y134); the Medical Research Foundation of
Guangdong Province (grant no. A2015620); and the Graduate Student
Research Innovation Project of Hunan Province (grant no.
CX2013B396).